Addressing Personal Protective Equipment (PPE) Decontamination: Methylene Blue and Light Inactivates SARS-CoV-2 on N95 Respirators and Masks with Maintenance of Integrity and Fit (preprint)

BackgroundThe coronavirus disease 2019 (COVID-19) pandemic has resulted in severe shortages of personal protective equipment (PPE) necessary to protect front-line healthcare personnel. These shortages underscore the urgent need for simple, efficient, and inexpensive methods to decontaminate SARS-CoV-2-exposed PPE enabling safe reuse of masks and respirators. Efficient decontamination must be available not only in low-resourced settings, but also in well-resourced settings affected by PPE shortages. Methylene blue (MB) photochemical treatment, hitherto with many clinical applications including those used to inactivate virus in plasma, presents a novel approach for widely applicable PPE decontamination. Dry heat (DH) treatment is another potential low-cost decontamination method. MethodsMB and light (MBL) and DH treatments were used to inactivate coronavirus on respirator and mask material. We tested three N95 filtering facepiece respirators (FFRs), two medical masks (MMs), and one cloth community mask (CM). FFR/MM/CM materials were inoculated with SARS-CoV-2 (a Betacoronavirus), murine hepatitis virus (MHV) (a Betacoronavirus), or porcine respiratory coronavirus (PRCV) (an Alphacoronavirus), and treated with 10 {micro}M MB followed by 50,000 lux of broad-spectrum light or 12,500 lux of red light for 30 minutes, or with 75{degrees}C DH for 60 minutes. In parallel, we tested respirator and mask integrity using several standard methods and compared to the FDA-authorized vaporized hydrogen peroxide plus ozone (VHP+O3) decontamination method. Intact FFRs/MMs/CM were subjected to five cycles of decontamination (5CD) to assess integrity using International Standardization Organization (ISO), American Society for Testing and Materials (ASTM) International, National Institute for Occupational Safety and Health (NIOSH), and Occupational Safety and Health Administration (OSHA) test methods. FindingsOverall, MBL robustly and consistently inactivated all three coronaviruses with at least a 4-log reduction. DH yielded similar results, with the exception of MHV, which was only reduced by 2-log after treatment. FFR/MM integrity was maintained for 5 cycles of MBL or DH treatment, whereas one FFR failed after 5 cycles of VHP+O3. Baseline performance for the CM was variable, but reduction of integrity was minimal. InterpretationMethylene blue with light and DH treatment decontaminated masks and respirators by inactivating three tested coronaviruses without compromising integrity through 5CD. MBL decontamination of masks is effective, low-cost and does not require specialized equipment, making it applicable in all-resource settings. These attractive features support the utilization and continued development of this novel PPE decontamination method.

Duplex formation between the template and the nascent strand in the transcription-regulating sequences determines the site of template switching in SARS - CoV-2 (preprint)

Recently published transcriptomic data of the SARS-CoV-2 coronavirus show that there is a large variation in the frequency and steady state levels of subgenomic mRNA sequences. This variation is derived from discontinuous subgenomic RNA synthesis where the polymerase switches template from a 3 proximal genome body sequence to a 5 untranslated leader sequence. This leads to a fusion between the common 5 leader sequence and a 3 proximal body sequence in the RNA product. This process revolves around a common core sequence (CS) that is present at both the template sites that make up the fusion junction. Base-pairing between the leader CS and the nascent complementary minus strand body CS, and flanking regions (together called the transcription regulating sequence, TRS) is vital for this template switching event. However, various factors can influence the site of template switching within the same TRS duplex. Here, we model the duplexes formed between the leader and complementary body TRS regions, hypothesising the role of the stability of the TRS duplex in determining the major sites of template switching for the most abundant mRNAs. We indicate that the stability of secondary structures and the speed of transcription play key roles in determining the probability of template switching in the production of subgenomic RNAs.

EVs analysis in the COVID-19 era: insights on serum inactivation protocols towards downstream isolation and analysis (preprint)

Since the outbreak of COVID-19 crisis, the handling of biological samples from confirmed or suspected SARS-CoV-2 positive individuals demanded the use of inactivation protocols to ensure laboratory operators safety. While not standardized, these practices can be roughly divided in two categories, namely heat inactivation and solvent-detergent treatments. As such, these routine procedures should also apply to samples intended for Extracellular Vesicles (EVs) analysis. Assessing the impact of virus inactivating pre-treatments is therefore of pivotal importance, given the well-known variability introduced by different pre-analytical steps on downstream EVs isolation and analysis. Arguably, shared guidelines on inactivation protocols tailored to best address EVs-specific requirements will be needed among the EVs community, yet deep investigations in this direction havent been reported so far. In the attempt of sparking interest on this highly relevant topic, we here provide preliminary insights on SARS-CoV-2 inactivation practices to be adopted prior serum EVs analysis by comparing solvent/detergent treatment vs. heat inactivation. Our analysis entailed the evaluation of EVs recovery and purity along with biochemical, biophysical and biomolecular profiling by means of Nanoparticle Tracking Analysis, Western Blotting, Atomic Force Microscopy, miRNA content (digital droplet PCR) and tetraspanin assessment by microarrays. Our data suggest an increase in ultracentrifugation (UC) recovery following heat-treatment, however accompanied by a marked enrichment in EVs-associated contaminants. On the contrary, solvent/detergent treatment is promising for small EVs (< 150 nm range), yet a depletion of larger vesicular entities was detected. This work represents a first step towards the identification of optimal serum inactivation protocols targeted to EVs analysis.

Decontamination of SARS-CoV-2 and other RNA viruses from N95 level meltblown polypropylene fabric using heat under different humidities (preprint)

In March of 2020, the World Health Organization declared a pandemic of coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The pandemic led to a shortage of N95-grade filtering facepiece respirators (FFRs), especially for protection of healthcare professionals against airborne transmission of SARS-CoV-2. We and others have previously reported promising decontamination methods that may be applied to the recycling and reuse of FFRs. In this study we tested disinfection of three viruses including SARS-CoV-2, dried on a piece of meltblown fabric, the principal component responsible for filtering of fine particles in N95-level FFRs, under a range of temperatures (60-95{degrees}C) at ambient or 100% relative humidity (RH) in conjunction with filtration efficiency testing. We found that heat treatments of 75{degrees}C for 30 min or 85{degrees}C for 20 min at 100% RH resulted in efficient decontamination from the fabric of SARS-CoV-2, human coronavirus NL63 (HCoV-NL63), and another enveloped RNA virus, chikungunya virus vaccine strain 181 (CHIKV-181), without lowering the meltblown fabrics filtration efficiency.

Is the fit of N95 facial masks effected by disinfection? A study of heat and UV disinfection methods using the OSHA protocol fit test. (preprint)

The current COVID-19 pandemic has highlighted global supply chain shortcomings in the US hospital delivery system, most notably personal protective equipment (PPE) and COVID-19 is found on these masks ~ 7 days. Recent work from our group has shown two promising disinfection methods for N95 facial masks, dry heat (hot air (75C, 30 min) and UVGI which is UVGI 254 nm, 8W, 30 min. Using N95 five models of N95 masks from three different manufacturers we determined the following: 1) Hot air treated N95 masks applied over 5 cycles did not degrade the fit of masks (1.5% change in fit factor, p = .67), 2) UVGI treated N95 masks applied over 10 cycles were significantly degraded in fit and did not pass quantitative fit testing using OSHA testing protocols on a human model (-77.4% change in fit factor, p = .0002).

Can N95 respirators be reused after disinfection? And for how many times? (preprint)

The Coronavirus Disease 2019 (COVID-19) pandemic has led to a major shortage of N95 respirators, which are essential to protecting healthcare professionals and the general public who may come into contact with the virus. Thus, it is essential to determine how we can reuse respirators and other personal protection in these urgent times. We investigated multiple commonly used and easily deployable, scalable disinfection schemes on media with particle filtration efficiency of 95%. Among these, heating ([≤]85 {degrees}C) under various humidities ([≤]100% RH) was the most promising, nondestructive method for the preservation of filtration properties in meltblown fabrics as well as N95-grade respirators. Heating can be applied up to 50 cycles (85 {degrees}C, 30% RH) without observation in the degradation of meltblown filtration performance. Ultraviolet (UV) irradiation was a secondary choice which was able to withstand 10 cycles of treatment and showed small degradation by 20 cycles. However, UV can also potentially impact the material strength and fit of respirators. Finally, treatments involving liquids and vapors require caution, as steam, alcohol, and household bleach may all lead to degradation of the filtration efficiency, leaving the user vulnerable to the viral aerosols.